Glutamate receptors mediate a slow Ca2+-permeable component of synaptic transmission, and are comprised of three classes: AMPA, kainate, and NMDA receptors. A great deal of information about how NMDA receptors are assembled, trafficked, and function has been derived primarily from work with recombinant tetrameric NMDA-Rs comprised of two identical GluN1 subunits and two identical GluN2 subunits. However, a large fraction of NMDA-Rs in the adult brain are triheteromeric receptors that contain GluN1 plus two different GluN2 subunits (e.g. GluN1/GluN2A/GluN2B). The wealth of functional and structural data describing NMDA-Rs with two identical GluN2 subunits is in stark contrast to a lack of even minimal understanding of the function and pharmacology of receptors that contain 2 different GluN2 subunits. Because highly divergent properties are imparted by different GluN2 subunits, it is impossible to predict the behavior or pharmacology of a receptor that contains two different GluN2 subunits. The question of how NMDA-R function is impacted by two different GluN2 subunits is also relevant for a growing number of apparent disease-causing human NMDA-R mutations (particularly in GluN2A). Heterozygous patients will have a substantial number of receptors with a single copy of the mutant subunit. Because NMDA-Rs with a single mutant subunit will function differently than those with zero or two copies, it is imperative to understand their function and sensitivity to candidate therapies. In order to understand the triheteromeric NMDA-Rs prevalent in native systems and disease, we propose to explore the functional and pharmacological properties of NMDA-Rs that contain two different GluN2 subunits or 0,1,2 copies of disease- associated mutant GluN2 subunits. This will be accomplished using molecular methods developed during the previous funding cycle that allow control of which GluN2 subunits are incorporated into cell surface receptors. The proposed experiments address three aims: Aim 1. What functional properties dominate when two GluN2 subunits reside in a single NMDA-R complex? We will determine which subunit controls the agonist potency, response time course, channel properties, and pharmacological sensitivity of triheteromeric receptors containing two different GluN2 subunits. Aim 2. Does GluN2C require co-assembly with GluN2A or GluN2B for surface expression? We will use new pharmacological tools that are sensitive to GluN2 stoichiometry and evaluation of single channel properties to examine whether receptors with two GluN2C subunits reach the plasma membrane in GluN2C-expressing olfactory neurons, spinal neurons, and adrenergic neurons in locus coeruleus. Aim 3. How do 0, 1, 2 copies of disease-associated human mutations influence NMDA-R function? We will assess glutamate and glycine EC50 in Xenopus oocytes and single channel properties for mutations in transmembrane domain / linkers implicated in gating. Mutations near the extracellular end of the transmembrane helices enhance channel activation.